Viruses, drug delivery vectors, and other external particles exhibit a variety of complex behaviors on and in the cell membrane that are reflective of their physical and chemical properties, including size, shape, charge and the availability of membrane receptors, before they trigger internalization pathways to enter the cell. Understanding the dynamics of these cellular membrane processes is essential for many important human health related problems, such as the rational design of nanoparticle-based drug delivery systems and the prevention and control of infectious pathogens. The past efforts provided excellent visualization of cellular membrane processes, but primarily for translational dynamics. This proposal focuses on utilizing the recently-developed single particle orientation and rotational tracking (SPORT) technique to elucidate the characteristic live-cell rotational dynamics. SPORT affords high spatial, angular, and temporal resolutions simultaneously, for visualizing the rotational dynamics of anisotropic plasmonic gold nanorods in live cells in differential interference contrast (DIC) microscopy. By using SPORT, the proposed research will acquire new fundamental knowledge about the detailed rotational dynamics of cellular membrane processes, such as adhesion, transport, and endocytosis of functionalized nanoparticles, as may be relevant to drug delivery and viral entry. The rotational patterns on cell membranes for functionalized gold nanorods will be identified and correlated with their lateral movements and the presence of relevant functional biomolecules tagged with fluorescent proteins. The characteristic rotational motions of cargos during different internalization pathways will also be visualized directly, leading to new opportunities for understanding the timing, signaling and chemical and mechanical functions of protein modules involved in different pathways. Computer simulations will be developed to understand the effects of nanoparticle shapes, sizes and surface modifiers. The simulations will aid the project by providing suggestions for further informative experiments. Finally, SPORT will be utilized to study the uptake mechanism of aptamer-loaded gold nanostars in cancer cells. The proposed research may initiate a shift in the current research paradigm on membrane structure and function by demonstrating the importance of rotational dynamics at the single molecule and nanoparticle level. A thorough understanding of the fundamental motions in evidence will inform about the details of the molecular mechanisms involved in the diffusion of membrane proteins and parallel internalization pathways that will be critical for the better design of antiviral drugs, as well asthe development of targeted delivery vehicles and anti-cancer medicines.